2型登革热病毒蛋白酶NS2B-NS3p活性构象的晶体结构研究

Dengue is an acute febrile disease caused by dengue viruses and is becoming a continuing global threat. However, there are currently no available drugs and no licensed vaccines. Dengue virus protease NS2B-NS3 is a typical trypsin like serine protease, and plays a vital role in the process of virus maturation with processing the poly-protein precursor. Therefore it is widely thought as a potential target of antivirus. The crystal structure of DENV2 NS2B-NS3p in inactive conformation was resolved. However, it is little known about the molecular mechanism of proteolytic processing till now. Thus there is no reliable structure to guide the effective antivirus drug development against NS2B-NS3p. In this study, we aim to determine the crystal structure of DENV2 NS2B-NS3p in active conformation. Coupled with the inactive crystal structure of DENV2 NS2B-NS3p, the active crystal structure of DENV3 NS2B-NS3p will elucidate the catalytic mechanism of this enzyme. This will pave the way for the design of antiviral drug against DENV2 NS2B-NS3p. In our preliminary work, the active two-component protease in an unlinked form was co-expressed. NMR analysis showed that the unlinked NS2B-NS3p mainly adopted closed active conformation in solution. Furthermore, we have obtained the crystal of the unlinked NS2B-NS3p. These data will greatly facilitate our proposed structural study of this enzyme detailed in this proposal.

登革热是感染登革热病毒引起的急性传染病，已成为全球性的健康威胁，却没有有效的防治策略。登革热病毒蛋白酶NS2B-NS3p属于丝氨酸蛋白酶，因酶解切割病毒蛋白前体多肽链而在病毒生命周期中扮演着关键作用，被广泛看作潜在的抗病毒靶标。NS2B-NS3p非活性构象的晶体结构已有报导，但无法解释蛋白酶解机制，也不能有效地用于基于结构的抗病毒药物研发。本项目旨在通过晶体学方法解析DENV2 NS2B-NS3p活性构象的结构，再通过结构比较分析，阐释其酶催化机理，为以该蛋白为靶标的抗登革热病毒药物研发夯实理论基础。申请人在前期工作中获得了新型的、具有蛋白酶活性的、共表达形式的NS2B-NS3p（unlinked NS2B-NS3p），并通过核磁研究表明unlinked NS2B-NS3p在溶液中主要采取活性构象，且已获得了其晶体生长条件，为本项目的顺利开展打下了扎实的基础。